In microbiology, microorganisms are classified in taxonomic categories, nomenclature being used to name the units delineated and characterised by classification. Identification by classical procedures involves the use of criteria established for classification and nomenclature in order to identify microorganisms by comparing the characteristics of an unknown unit with known units. Thus with a newly isolated microorganism, its identification requires an adequate characterisation thereof and then a comparison with published descriptions of other similar microorganisms.
To identify an organism of interest that is present in a specimen of mineral, plant or animal origin, it is first necessary to obtain an isolated colony of the microorganism. There are well developed procedures for growing or cultivating microorganisms in the laboratory on nutrient material, some of these procedures requiring special conditions such as the absence of free oxygen. By incubating a nutrient agar-type medium, using the streak-plate or pour-plate method, cells are individually separated. In incubation, individual cells reproduce rapidly to generate a visible colony of cells, each colony being a pure sample of a single kind of microorganism.
In order to identify an unknown cell, classical techniques call for the use of high-magnification optical or electron microscopes in order to determine colony and cell morphology. In addition numerous other characteristics may have to be determined, including staining characteristics, susceptibility to antimetabolites and serological and biochemical properties. The procedures for the identification of bacteria are set forth in detail in chapter 5 of the text "Clinical Bacteriology"--Fifth Ed.--J. Stokes et al., published by Arnold (London) 1980.
Thus, the identification of microorganisms by classical techniques is time-consuming, labour-intensive and expensive and, not withstanding the high order of technical skill required, is liable to error.
The identification of microorganisms to clearly of great importance in the medical and veterinary fields. However, in recent years the need for efficient and relatively rapid identification techniques has become even more pressing owing to the remarkable expansion of environmental and industrial microbiology. Thus, the cultivation of microorganisms in food processing, in the fermentation of alcoholic beverages, and in the manufacture of pharmaceuticals and of such industrial reagents as the alcohols and acetic acid is already well established. The use of microorganisms has been proposed not only for syntheses but also for counter-pollution measures; an interesting use of selected microorganisms to degrade products of industrial organic syntheses is described in GB-A-2010327. Furthermore, genetic engineering is expected markedly to increase the range of applications of microbiology in industry and agriculture (see Scientific American, September 1981).
There have, of course, been attempts to improve upon the classical techniques for microorganism identification. For example, Tsukamura and Mizuno (Kekkaku 1980, 55 (12) pages 525-530) disclose a method by which certain selected microorganisms can be distinguished. The precultured organism was incubated for 24 hours in a reaction medium containing L-.sup.35 S-methionine, after which the cells were centrifuged, washed and extracted with ethyl ether/ethanol. The extracted material was subjected to further extraction using petroleum ether and the resultant material was subjected to thin-layer chromatography. Any radioactive spots in the thin-layer were detected by an automatic scannner. These Japanese workers were able to distinguish Mycobacterium nonchromogenicum (which gave one strong radioactive spot at an Rf value 0.70-0.80) from M.terrae and M.triviale (which two organisms produced no spot or only a trace spot). They were also able to differentiate between two particular Rhodococcus species and to distinguish Rhodococcus species from Nocardia species (the former giving a spot at Rf 0.10 or Rf 0.95, whereas Nocardia displayed no such spots).
The method described in the Kekkaku article clearly does not qualify as a general method for the identification of microorganisms: thus, the Japanese workers were unable to differentiate M.terrae and M.triviale. Furthermore, the method required an initially high concentration of microorganism and a long incubation period in the .sup.35 S-methionine-containing medium. An interesting observation is that, although methionine is an amino acid (such acids being the building blocks of proteins), the petroleum ether extraction would not have taken up proteins and thin-layer chromatography (TLC) is not a useful technique for resolving proteins. It would appear, therefore, that any radioactive spot that may be detected in the thin layer is not due to the incorporation of the .sup.35 S-methionine in a protein product of the metabolism of the organism, but is due to a product of a secondary reaction between the radioactive label and a compound derived from the organism.
With a view to automating the identification of microorganisms, U.S. Pat. No. 4,288,543 to Sielaff et al. discloses a procedure in which the susceptibility of various strains of bacteria to antimicrobial agents is tested, this being done in conjunction with a determination of the light-scattering index of the organism being tested. The numerical growth data obtained by the light scatter comparisons are analysed by computer-assisted statistical techniques in order to identify the organism. The admitted drawback to this procedure is that one should use agents not in common therapeutic use in order to avoid errors resulting from strains that have become immune to various therapeutically utilised antibiotic agents. Furthermore, it is necessary to divide the initial sample of the microorganism (specifically a bacterium) into a number of sub-samples, each of which has to be inoculated with a respective growth-inhibiting agent, incubated and then tested. The Sielaff patent also makes of record other publications dealing with the automated identification of bacteria by computer analysis of growth inhibition patterns.
The logical approach to the problem of identification is to find or create an indentifier, namely a characteristic by means of which the identity of an unknown can be determined. Thus, fingerprints are regarded as identifiers for human beings, since a person can be identified by his fingerprints alone, without reference to that person's other characteristics, such as sex, age, height, weight, shape, eye colour and the like. However, a problem in applying this approach to microbiology is the difficulty of selecting a microorganism characteristic that really is an identifier, that can be routinely utilised as such, and that is applicable throughout the group of microorganisms (especially bacteria) in question. The identifier, like a fingerprint, should be substantially universal. Obviously, like human fingerprints, there may be exceptions, but the generation of the identifier should be the rule and not the exception.
One attempt to tackle this problem is described in GB-A-1489255. That specification describes a process for the identification of a microorganism which comprises inoculating a plurality of different .sup.14 C-labelled substrates with an unknown organism and incubating the substrates for a time sufficient to cause metabolic breakdown of at least some of the substrates by the organism to produce the radioactive gas .sup.14 CO.sub.2. The gas that is evolved is analysed for radioactivity in order to obtain a "substrate radiorespirometric profile" for the unknown microorganism. Such a profile is said (page 3, lines 3-13) to serve as a fingerprint of the unknown microorganism, in that the profile can be compared to standard profiles obtained in the same manner from known microbes. That technique requires the unknown microorganism to be tested against a sufficient number of substrates taken individually in order to obtain a meaningful profile; for instance, in the specific Example of GB-A-1489255, thirty substrates are used. Thus, each unknown is subjected, in effect, to a series of complex tests and this must render it difficult to standardise the test procedure.